In the process of infecting plants, plant parasitic nematodes release a series of proteins that play an essential role in the successful infection and pathogenesis of plant cells and tissues through stylets or body wa...In the process of infecting plants, plant parasitic nematodes release a series of proteins that play an essential role in the successful infection and pathogenesis of plant cells and tissues through stylets or body walls. In this study,based on transcriptome data, a chorismate mutase gene of Radopholus similis(RsCM) was identified and cloned,which is a single copy gene specifically expressed in the oesophageal gland and highly expressed in juveniles and females. Transient expression of RsCM in tobacco leaves showed that it was localised in the cytoplasm and nucleus of tobacco leaf cells, which inhibited the pattern-triggered immunity(PTI) induced by flg22, including callose deposition and defence gene expression, and cell death induced by immune elicitors BAX, but could not inhibit cell death induced by immune elicitors Gpa2/RBP-1. The RNA interference(RNAi) transgenic tomato of RsCM obviously inhibited the infection, pathogenicity, and reproduction of R. similis. However, the resistance of the overexpression transgenic tomato of RsCM to R. similis infection was significantly reduced, and the expression levels of two salicylic acid(SA) pathway genes(PR1 and PR5) in roots infected by the nematode were significantly down-regulated,which indicated that RsCM might be involved in the inhibition of SA pathway. The results of this study demonstrate that RsCM suppresses the host immune system and might be a new target for the control of R. similis, which also provides new data for the function and mechanism of CM genes of migratory parasitic plant nematodes.展开更多
Vitamin B 12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes,which have been grouped into three subfamilies depending on their cofactors.Among them,methylmal...Vitamin B 12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes,which have been grouped into three subfamilies depending on their cofactors.Among them,methylmalonyl-CoA mutase (MCM) has been extensively studied.This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor participating in the generation of radicals that allow isomerization of the substrate.The crystal structure of MCM determined in Propionibacterium freudenreichii var.shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme,coenzyme,and substrate.The existence of human methylmalonic acidemia (MMA) due to the presence of mutations in MCM shows the importance of its role in metabolism.The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein,there are significant differences in the structural organization of the two proteins.Recent studies have identified the involvement of an accessory protein called MMAA,which interacts with MCM to prevent MCM's inactivation or acts as a chaperone to promote regeneration of inactivated enzyme.The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase,the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans.It is still necessary to study the mechanisms involved in more detail using new methods.展开更多
BACKGROUND Drugs targeting mitochondria can induce mitophagy and restrain proliferation in colorectal cancer(CRC)cells.Phosphoglycerate mutase family member 5(PGAM5)activates serine/threonine PTEN-induced putative kin...BACKGROUND Drugs targeting mitochondria can induce mitophagy and restrain proliferation in colorectal cancer(CRC)cells.Phosphoglycerate mutase family member 5(PGAM5)activates serine/threonine PTEN-induced putative kinase 1/Parkin pathway-mediated mitophagy.However,there are few studies on the clinical and prognostic significance of expression of PGAM5 protein and mitophagy-related protein Parkin in patients.AIM To assess the clinical significance of PGAM5 and Parkin proteins,as biomarkers for diagnosis and prognosis of CRC,by studying their expression in advanced CRC tissues and their association with clinicopathological parameters.METHODS The expression of PGAM5 and Parkin in CRC tissues from 100 patients was determined by immunohistochemistry.Each case was evaluated by using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-4).The final staining score was calculated as the intensity score multiplied by the proportion score.Specimens were categorized as either high or low expression according to the Youden index,and the association between the expression of PGAM5 or Parkin and clinicopathological factors was ascertained.Additionally,we employed western blot to measure PGAM5 and Parkin protein expression in six matched pairs of CRC and adjacent non-tumor tissues.RESULTS Immunohistochemical and western blot findings showed that both PGAM5 and Parkin protein expression in tumor tissues was significantly higher than that in the adjacent tissues:PGAM5 and Parkin were mainly expressed in the cytoplasm of colonic epithelial cells.PGAM5 and Parkin protein levels were significantly positively correlated in advanced CRC tissues.Moreover,reduced Parkin protein expression was an independent prognostic factor for overall survival and progression-free survival in CRC patients as evinced by multivariate analysis.CONCLUSION The expression of PGAM5 protein and mitophagy-related protein Parkin has diagnostic significance for CRC and may become new biomarkers.Parkin may be a potential marker for the survival of CRC patients.展开更多
<p style="margin-left:10.0pt;"> <br /> </p> <p style="margin-left:10.0pt;"> <span>The coenzyme B<sub>12</sub> dependent glutamate mutase is composed of two...<p style="margin-left:10.0pt;"> <br /> </p> <p style="margin-left:10.0pt;"> <span>The coenzyme B<sub>12</sub> dependent glutamate mutase is composed of two apoenzyme proteins subunits;S and E<sub>2</sub>, which while either fused or separate assemble with coenzyme B<sub>12</sub> to form an active holoenzyme (E<sub>2</sub>S<sub>2</sub>-B<sub>12</sub>) for catalyzing the reversible isomerization between (<i>S</i>)-glutamate and (2<i>S</i>, 3<i>S</i>)-3-methylas</span><span>- </span><span>partate. In order to assay the activity of glutamate mutase by UV spectrophotometry, this reaction is often coupled with methylaspartase which deaminates (2<i>S</i>, 3<i>S</i>)-3-methylaspartate to form mesaconate (<i>λ</i><sub>max</sub> = 240 nm, </span><span>Ɛ</span><sub><span>240</span></sub><span> = 3.8 mM<sup>-1</sup>·cm<sup>-1</sup>). The activities of different reconstitutions of glutamate mu<span>tase from separate apoenzyme components S and E in varied amount</span></span><span>s</span><span> of </span><span>coenzyme B<sub>12</sub> and adenosylpeptide B<sub>12</sub> as cofactors were measured by this assay and used to reveal the binding properties of the cofactor by the Michaelis</span><span>- </span><span>Menten Method. The values of <i>K<sub>m</sub></i> for coenzyme B<sub>12</sub> in due to reconstitutions of holoenzyme in 2, 7 and 14 S: E were determined as;1.12 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B<sub>12</sub>;1.07 ± 0.04 μM and 0.35 ± 0.05 μM as obtained from respective 2 and 14 S: E compositions of holoenzyme. Analysis of these kinetics results curiously as<span>sociate</span></span><span>s</span><span> the increasing affinity of cofactors to apoenzyme with</span><span> </span><span>increased amount of component S used in reconstituting holoenzyme from separate</span><span> apoenzyme components and cofactor.</span><span> Moreover, in these studies a new method for assaying the activity of glutamate mutase was developed, whereby glutamate mutase activity is measured via depletion of NADH (<i>λ</i><sub>max</sub> = 340 nm, </span><span>Ɛ</span><sub><span>340</span></sub><span> = 6.3 mM<sup>-1</sup>·cm<sup>-1</sup>) as determined by UV spectrophotometry after addition of (2<i>S</i>,<span> 3<i>S</i>)-3-methylaspartate and pyruvate to a mixture of E<sub>2</sub>S<sub>2</sub>-B<sub>12</sub> and two auxiliary </span><span>holoenzymes system;pyridoxal-5-phosphate dependent glutamate-pyruvate </span><span>aminotransferase and N</span>ADH dependent (<i>R</i>)-2-hydroxyglutarate dehydrogenas<span>e. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (<i>S</i>)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (<i>R</i>)-2-hydroxylglutarate</span> dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2<i>S</i>, 3<i>S</i>)-3-methylaspartate in the reaction of glutamate mutase as <i>K</i><sub>m</sub>= 7 ± 0.07 mM and <i>k</i><sub>cat</sub>= 0.54 ± 0.6 s<sup>-1</sup>. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, <i>K</i><sub>eq</sub> = [(<i>S</i>)-glutamate] × [(2<i>S</i>, 3<i>S</i>)-3-methylaspartate]<sup>-1</sup> = 16, where the kinetic constants of (<i>S</i>)-glutamate were determined by the standard methylaspartase coupled assay.<span></span></span> </p> <p> <br /> </p>展开更多
Mesaconic acid has a special chemical structure and can undergo a series of reactions such as polymerization and addition. It is an important chemical intermediate and widely used in material, chemical and other indus...Mesaconic acid has a special chemical structure and can undergo a series of reactions such as polymerization and addition. It is an important chemical intermediate and widely used in material, chemical and other industries. The chemical synthesis of mesaconic acid requires nitric acid, which is dangerous and harmful to the environment. The production of mesaconic acid by microbial fermentation has the characteristics of low raw material price, high efficiency and strong specificity, and thus a strong industrial application prospect. Mesaconic acid is an intermediate product of glutamic acid degradation pathway of microorganisms such as Clostridium tetani. However, at present, few reports have been conducted on the production of mesaconic acid by metabolic engineering microorganisms. In this study, glutamate mutase(GLM) and 3-methylaspartate ammonialyase(MAL) from C. tetani were recombined and expressed in Escherichia coli, and the obtained strain, BL21(DE3)/pETDuet-1-MAL-mutS-mutE, achieved the yield of mesaconic acid of 1.06 g/L. Compared with the wild type, the yields of mesaconic acid from mutants G133A and G133S increased by 21% and 16%, respectively. After 24 h of flask fermentation, the yields of mesaconic acid reached 1.28 and 1.23 g/L, respectively. This study can provide reference for microbial synthesis of mesaconic acid.展开更多
基金funded by the Guangdong Basic and Applied Basic Research Foundation,China(2021A1515011273)he National Natural Science Foundation of China(31071665)。
文摘In the process of infecting plants, plant parasitic nematodes release a series of proteins that play an essential role in the successful infection and pathogenesis of plant cells and tissues through stylets or body walls. In this study,based on transcriptome data, a chorismate mutase gene of Radopholus similis(RsCM) was identified and cloned,which is a single copy gene specifically expressed in the oesophageal gland and highly expressed in juveniles and females. Transient expression of RsCM in tobacco leaves showed that it was localised in the cytoplasm and nucleus of tobacco leaf cells, which inhibited the pattern-triggered immunity(PTI) induced by flg22, including callose deposition and defence gene expression, and cell death induced by immune elicitors BAX, but could not inhibit cell death induced by immune elicitors Gpa2/RBP-1. The RNA interference(RNAi) transgenic tomato of RsCM obviously inhibited the infection, pathogenicity, and reproduction of R. similis. However, the resistance of the overexpression transgenic tomato of RsCM to R. similis infection was significantly reduced, and the expression levels of two salicylic acid(SA) pathway genes(PR1 and PR5) in roots infected by the nematode were significantly down-regulated,which indicated that RsCM might be involved in the inhibition of SA pathway. The results of this study demonstrate that RsCM suppresses the host immune system and might be a new target for the control of R. similis, which also provides new data for the function and mechanism of CM genes of migratory parasitic plant nematodes.
基金Project (No.IN208411) partially supported by the PAPIIT-UNAM of Mexico
文摘Vitamin B 12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes,which have been grouped into three subfamilies depending on their cofactors.Among them,methylmalonyl-CoA mutase (MCM) has been extensively studied.This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor participating in the generation of radicals that allow isomerization of the substrate.The crystal structure of MCM determined in Propionibacterium freudenreichii var.shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme,coenzyme,and substrate.The existence of human methylmalonic acidemia (MMA) due to the presence of mutations in MCM shows the importance of its role in metabolism.The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein,there are significant differences in the structural organization of the two proteins.Recent studies have identified the involvement of an accessory protein called MMAA,which interacts with MCM to prevent MCM's inactivation or acts as a chaperone to promote regeneration of inactivated enzyme.The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase,the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans.It is still necessary to study the mechanisms involved in more detail using new methods.
基金Supported by the Natural Science Foundation of Liaoning Province,No.2019-BS-279.
文摘BACKGROUND Drugs targeting mitochondria can induce mitophagy and restrain proliferation in colorectal cancer(CRC)cells.Phosphoglycerate mutase family member 5(PGAM5)activates serine/threonine PTEN-induced putative kinase 1/Parkin pathway-mediated mitophagy.However,there are few studies on the clinical and prognostic significance of expression of PGAM5 protein and mitophagy-related protein Parkin in patients.AIM To assess the clinical significance of PGAM5 and Parkin proteins,as biomarkers for diagnosis and prognosis of CRC,by studying their expression in advanced CRC tissues and their association with clinicopathological parameters.METHODS The expression of PGAM5 and Parkin in CRC tissues from 100 patients was determined by immunohistochemistry.Each case was evaluated by using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-4).The final staining score was calculated as the intensity score multiplied by the proportion score.Specimens were categorized as either high or low expression according to the Youden index,and the association between the expression of PGAM5 or Parkin and clinicopathological factors was ascertained.Additionally,we employed western blot to measure PGAM5 and Parkin protein expression in six matched pairs of CRC and adjacent non-tumor tissues.RESULTS Immunohistochemical and western blot findings showed that both PGAM5 and Parkin protein expression in tumor tissues was significantly higher than that in the adjacent tissues:PGAM5 and Parkin were mainly expressed in the cytoplasm of colonic epithelial cells.PGAM5 and Parkin protein levels were significantly positively correlated in advanced CRC tissues.Moreover,reduced Parkin protein expression was an independent prognostic factor for overall survival and progression-free survival in CRC patients as evinced by multivariate analysis.CONCLUSION The expression of PGAM5 protein and mitophagy-related protein Parkin has diagnostic significance for CRC and may become new biomarkers.Parkin may be a potential marker for the survival of CRC patients.
文摘<p style="margin-left:10.0pt;"> <br /> </p> <p style="margin-left:10.0pt;"> <span>The coenzyme B<sub>12</sub> dependent glutamate mutase is composed of two apoenzyme proteins subunits;S and E<sub>2</sub>, which while either fused or separate assemble with coenzyme B<sub>12</sub> to form an active holoenzyme (E<sub>2</sub>S<sub>2</sub>-B<sub>12</sub>) for catalyzing the reversible isomerization between (<i>S</i>)-glutamate and (2<i>S</i>, 3<i>S</i>)-3-methylas</span><span>- </span><span>partate. In order to assay the activity of glutamate mutase by UV spectrophotometry, this reaction is often coupled with methylaspartase which deaminates (2<i>S</i>, 3<i>S</i>)-3-methylaspartate to form mesaconate (<i>λ</i><sub>max</sub> = 240 nm, </span><span>Ɛ</span><sub><span>240</span></sub><span> = 3.8 mM<sup>-1</sup>·cm<sup>-1</sup>). The activities of different reconstitutions of glutamate mu<span>tase from separate apoenzyme components S and E in varied amount</span></span><span>s</span><span> of </span><span>coenzyme B<sub>12</sub> and adenosylpeptide B<sub>12</sub> as cofactors were measured by this assay and used to reveal the binding properties of the cofactor by the Michaelis</span><span>- </span><span>Menten Method. The values of <i>K<sub>m</sub></i> for coenzyme B<sub>12</sub> in due to reconstitutions of holoenzyme in 2, 7 and 14 S: E were determined as;1.12 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B<sub>12</sub>;1.07 ± 0.04 μM and 0.35 ± 0.05 μM as obtained from respective 2 and 14 S: E compositions of holoenzyme. Analysis of these kinetics results curiously as<span>sociate</span></span><span>s</span><span> the increasing affinity of cofactors to apoenzyme with</span><span> </span><span>increased amount of component S used in reconstituting holoenzyme from separate</span><span> apoenzyme components and cofactor.</span><span> Moreover, in these studies a new method for assaying the activity of glutamate mutase was developed, whereby glutamate mutase activity is measured via depletion of NADH (<i>λ</i><sub>max</sub> = 340 nm, </span><span>Ɛ</span><sub><span>340</span></sub><span> = 6.3 mM<sup>-1</sup>·cm<sup>-1</sup>) as determined by UV spectrophotometry after addition of (2<i>S</i>,<span> 3<i>S</i>)-3-methylaspartate and pyruvate to a mixture of E<sub>2</sub>S<sub>2</sub>-B<sub>12</sub> and two auxiliary </span><span>holoenzymes system;pyridoxal-5-phosphate dependent glutamate-pyruvate </span><span>aminotransferase and N</span>ADH dependent (<i>R</i>)-2-hydroxyglutarate dehydrogenas<span>e. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (<i>S</i>)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (<i>R</i>)-2-hydroxylglutarate</span> dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2<i>S</i>, 3<i>S</i>)-3-methylaspartate in the reaction of glutamate mutase as <i>K</i><sub>m</sub>= 7 ± 0.07 mM and <i>k</i><sub>cat</sub>= 0.54 ± 0.6 s<sup>-1</sup>. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, <i>K</i><sub>eq</sub> = [(<i>S</i>)-glutamate] × [(2<i>S</i>, 3<i>S</i>)-3-methylaspartate]<sup>-1</sup> = 16, where the kinetic constants of (<i>S</i>)-glutamate were determined by the standard methylaspartase coupled assay.<span></span></span> </p> <p> <br /> </p>
文摘Mesaconic acid has a special chemical structure and can undergo a series of reactions such as polymerization and addition. It is an important chemical intermediate and widely used in material, chemical and other industries. The chemical synthesis of mesaconic acid requires nitric acid, which is dangerous and harmful to the environment. The production of mesaconic acid by microbial fermentation has the characteristics of low raw material price, high efficiency and strong specificity, and thus a strong industrial application prospect. Mesaconic acid is an intermediate product of glutamic acid degradation pathway of microorganisms such as Clostridium tetani. However, at present, few reports have been conducted on the production of mesaconic acid by metabolic engineering microorganisms. In this study, glutamate mutase(GLM) and 3-methylaspartate ammonialyase(MAL) from C. tetani were recombined and expressed in Escherichia coli, and the obtained strain, BL21(DE3)/pETDuet-1-MAL-mutS-mutE, achieved the yield of mesaconic acid of 1.06 g/L. Compared with the wild type, the yields of mesaconic acid from mutants G133A and G133S increased by 21% and 16%, respectively. After 24 h of flask fermentation, the yields of mesaconic acid reached 1.28 and 1.23 g/L, respectively. This study can provide reference for microbial synthesis of mesaconic acid.